The use of a light microscope paved the way to discovering many pathogenic microorganisms causing human diseases, advancing our knowledge of medicine. Without staining with a dye, Alphonse Laveran first noticed an unknown microorganism with actively mobile filaments in blood taken from an infected soldier under a microscope, later confirming it as a
Plasmodium male gamete with moving flagella [
7]. Once the Giemsa solution, a type of Romanowsky dye, was deployed to stain human blood smears on glass slides, scientists were able to identify
Plasmodium-infected erythrocytes under a light microscope. Thus, enumerating the parasitized erythrocytes and discriminating intraerythrocytic stages allows an estimation of the number of infected host cells (parasitaemia) and a measurement of parasite growth. Since then, Giemsa-based microscopy has become the gold standard for the diagnosis and clinical management of malaria [
8] and an essential part of anti-malarial drug susceptibility tests.
To assess the effect of a drug on the intraerythrocytic growth of malaria parasites, cultures of
Plasmodium spp. have been widely used as a malaria model in the preclinical phase of anti-malarial drug development and as a tool for drug resistance surveillance. To model malaria
in vitro, laboratory-adapted
P. falciparum strains or field-isolated strains are cultured with human erythrocytes, mostly those of blood group O, in RPMI 1640 medium supplemented with HEPES, sodium bicarbonate, and heat-inactivated human AB serum. The
in vitro or
ex vivo cultured
Plasmodium parasites are then incubated with serial dilutions of drug and subjected to enumerating parasitized erythrocytes (schizonts) using a microscope. Giemsa-based light microscopy is the main method used to measure the intraerythrocytic development of
Plasmodium parasites [
9,
10], and it has a limit of detection of 0.001% parasitaemia based on examination of thick blood film in routine microscopic diagnosis [
11]. In
Plasmodium growth inhibition assays, a synchronous culture is treated with a drug [
12]. Then, the development of
Plasmodium parasites is examined. There are several methods for the assessment of intraerythrocytic
Plasmodium growth: morphological observation under a microscope and biochemical measurements. For morphology-based assays, the microtechnique developed by Rieckmann et al. [
3] is simple and reliable and has still been used in recent reports [
13‐
18]. For biochemical assays, the microtechnique was modified to a semiautomatic tritiated hypoxanthine incorporation assay, measuring
Plasmodium uptake of radiolabelled hypoxanthine, a nucleic acid precursor, to assess the growth inhibitory effect of anti-malarial drugs [
19] or immune serum [
20]. Given a rapid, more precise and quantitative method, many recent reports have been adopted for the assessment of
Plasmodium growth inhibition in experiments [
21] and in a clinical trial setting [
22] and for surveillance of drug resistance in field isolates [
23]. In addition to DNA synthesis-based assays,
Plasmodium parasites in intraerythrocytic stages also actively synthesize cell membranes composed of phospholipids; thus, an assay based on incorporation of the radioisotope-labeled phospholipid precursor ethanolamine was developed [
24], allowing an assessment of anti-malarial compounds targeting enzyme functioning in fatty acid biosynthesis [
25]. Apart from biosynthesis-based tests, measurements of
Plasmodium-specific lactate dehydrogenase [
26‐
28] and histidine-rich protein II [
29,
30] are also available and have been widely used for anti-malarial drug tests [
31,
32].
In 1979, Howard et al. [
33] were the first to use the nucleic acid-binding fluorescent Hoechst 33,258 dye to detect
P. berghei-infected mouse erythrocytes. In early 1990, a fluorescence-based flow cytometer was introduced for drug susceptibility testing and parasite detection in human blood [
34,
35]. Compared to the gold standard Giemsa-based microscope, fluorescence-based flow cytometry consumes a relatively shorter period of time. Prior to microscopic examination, thick blood film preparation and Giemsa staining were required. Then, a well-trained microscopist enumerates
Plasmodium-infected cells and leukocytes. Approximately 16–20 h of drug testing was performed in a 96-well plate, while fluorescence-based flow cytometry consisting of cell staining, washing and acquisition was performed within 2 h [
36]. Despite its high sensitivity, reliability and applicability for high-throughput experiments, fluorescence-based flow cytometry is expensive. Large, complex flow cytometers have been transformed into a small, transportable, user friendly and low-cost format, providing a platform suitable for field settings [
36‐
38]. Thus, the principles of fluorochrome-based flow cytometry and the common processes that occur before and after flow cytometric assays are next briefly explained.